The role of DNA as a stable repository for genetic information is constantly challenged by the chemical reactivity of the DNA bases (Lindahl (1993) Nature 362:709-715). DNA molecules, like all other biomolecules, can be damaged in numerous ways. Nearly all atoms of the DNA bases are subject to some form of hydrolysis, oxidative damage or alkylation. Moreover, spontaneous damage due to replication errors, deamination, depurination and oxidation is compounded in the real world by the additional effects of radiation and environmental chemicals. If left uncorrected, these lesions can be lethal to a cell.
The xe2x80x9cpathwayxe2x80x9d most commonly employed to remove incorrect bases (like uracil) or alkylated bases (like 3-methyladenine) or other damaged bases is called xe2x80x9cbase excision repairxe2x80x9d. Excision repair relies on the redundant information in the duplex to remove a damaged base or nucleotide and replace it with a normal base by using the complementary strand as a template. In base excision repair the removal of the lesion occurs in two steps: First, the damaged base is released by a DNA glycosylase, and then the abasic sugar (AP site) is excised by AP endonucleases. Each base excision reaction is of a limited substrate range because the DNA glycosylases that initiate the repair process are in intimate contact with the lesion during catalysis.
It is an object of the present invention to provide inhibitors of DNA glycosylases in order to, e.g., manipulate the DNA repair response of a cell.
One aspect of the present invention provides a DNA glycosylase inhibitor represented in the general formula (I), or a pharmaceutically acceptable salt thereof: 
wherein,
B is a nucleoside purine or pyrimidiine base, or a heterocyclic analog thereof,
X is O, N, S or CH2;
R1, independently for each occurence, is absent or is a hydrogen, or an amino protecting group;
R2 is a hydrogen, a nucleotide or oligonucleotide, a phosphoryl, a phosphonate, a phosphoramidate, a carbamate, a phosphorothioate, a phosphorodithioate, a hydroxyl blocking group, or as valence and stability permit, a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl, a thiocarbonyl, a ketyl, an aldehyde, an amino, an acylamino, an amido, an amidino, a cyano, a nitro, an azido, a sulfonyl, a sulfoxido, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a phosphoryl, a phosphonate, a phosphinate, xe2x80x94(CH2)mxe2x80x94R8, xe2x80x94(CH2)mxe2x80x94OH, xe2x80x94(CH2)mxe2x80x94O-lower alkyl, xe2x80x94(CH2)mxe2x80x94O-lower alkenyl, xe2x80x94(CH2)mxe2x80x94O xe2x80x94(CH2)nxe2x80x948, xe2x80x94(CH2)mxe2x80x94SH, xe2x80x94(CH2)mxe2x80x94S-lower alkenyl, xe2x80x94(CH2)mxe2x80x94S-lower alkenyl, xe2x80x94(CH2)mxe2x80x94Sxe2x80x94(CH2)nxe2x80x948, or a solid or polymeric support;
R3 is a hydrogen, a nucleotide or oligonucleotide, a phosphoryl, a phosphonate, a phosphoramidate, a carbamate, a phosphorothioate, a phosphorodithioate, a hydroxyl blocking group, or as valence and stability permit, a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl, a thiocarbonyl, a ketyl, an aldehyde, an amino, an acylamino, an amido, an amidino, a cyano, a nitro, an azido, a sulfonyl, a sulfoxido, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a phosphoryl, a phosphonate, a phosphinate, xe2x80x94(CH2)mxe2x80x94R8, xe2x80x94(CH2)mxe2x80x94OH, xe2x80x94(CH2)mxe2x80x94O-lower alkyl, xe2x80x94(CH2)mxe2x80x94O-lower alkenyl, xe2x80x94(CH2)mxe2x80x94Oxe2x80x94(CH2)nxe2x80x948, xe2x80x94(CH2)mxe2x80x94SH, xe2x80x94(CH2)mxe2x80x94S-lower alkenyl, xe2x80x94(CH2)mxe2x80x94S-lower alkenyl, xe2x80x94(CH2)mxe2x80x94Sxe2x80x94(CH2)nxe2x80x94R8, or a solid or polymeric support;
R4, R5, R6 and R7 are each, independently, as valence and stability permit, hydrogen, a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl, a thiocarbonyl, a ketyl, an aldehyde, an amino, an acylamino, an amido, an amidino, a cyano, a nitro, an azido, a sulfonyl, a sulfoxido, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a phosphoryl, a phosphonate, a phosphinate, xe2x80x94(CH2)mxe2x80x94R8, xe2x80x94(CH2)mxe2x80x94OH, xe2x80x94(CH2)mxe2x80x94O-lower alkyl, xe2x80x94(CH2)mxe2x80x94O-lower alkenyl, xe2x80x94(CH2)mxe2x80x94Oxe2x80x94(CH2)nxe2x80x94R8, xe2x80x94(CH2)mxe2x80x94SH, xe2x80x94(CH2)mxe2x80x94S-lower alkyl, xe2x80x94(CH2)mxe2x80x94Sxe2x80x94(CH2)nxe2x80x94R8;
R8 is, independently for each occurrence, a substituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl or heterocycle, a carbonyl, a sulfonyl or a phosphoryl;
p is zero, 1 or 2, and n and m are independently for each occurrence zero or an integer in the range of 1 to 6,
which compound inhibits an N-glycosidic activity of a DNA glycosylase.
In preferred embodiments, the subject inhibitor is represented in the general formula: 
For example, the inhibitor is represented in the general formula: 
The subject inhibitor can be incorporated as part of an oligonucleotide, e.g., R2 and/or R3 are one or more nucleotides (including analogs). For istance, the nucleic acid can be at least 4, 8, 10, 15, 20, 30 or more nucleotide units in length.
In preferred embodiments, the subject compounds inhibit a DNA glycosylase which is a member of the EC category EC 3.2.2.-. For instance, the DNA glycosylase may be, e.g., a purine nucleosidase (EC 3.2.2.1), an inosine nucleosidase (EC 3.2.2.2), a uridine nucleosidase/uracil deglycosylase (EC 3.2.2.3), a ribosylpyrimidine nucleosidase (EC 3.2.2.8), an inosinate nucleosidase (EC 3.2.2.12), a 1-methyladenosine nucleosidase (3.2.2.13), a dna-deoxyinosine glycosidase (EC 3.2.2.15), a methylthioadenosine nucleosidase (EC 3.2.2.16), a DNA-3-methyladenine glycosidase (I) (EC 3.2.2.20), a DNA-3-methyladenine glycosidase (II) (EC 3.2.2.21), and a formamidopyrimidine-DNA glycosidase (EC 3.2.2.23). In certain embodiments, the the DNA glycosylase is a mammalian DNA glycosylase, such as MYH, Mpg, 3Mg, Ung1 and Ung2. In other embodiments, the DNA glycosylase is a bacterial DNA glycosylase such as MutM, MutT, fpg4 and MutY. In yet other embodiments, the DNA glycosylase is a viral DNA glycosylase from, e.g., an Epstein Barr Virus or Herpes Simplex Virus.
Another aspect of the present invention pertains to pharmaceutical preparations including the subject DNA glycosylase and a pharmaceutically acceptable carrier.
Another aspect of the present invention provides a solid support (or soluble polymeric support) derivatized with the subject DNA glycosylase.
Still another aspect of the present invention relates to a method for decreasing the DNA repair activity of a a cell comprising contacting the cell with one or more of the subject DNA glycosylase inhibitors. For instance, the subject method can be used for increasing the sensitivity of a cell to DNA damaging agents. It can be carried out on cells in culture, e.g., the glycosylase inhibitor is provided as a cell culture additive. Alternatively, it can be used in the treatment of an animal, e.g., the glycosylase inhibitor is administered to the animal as a therapeutic composition. In the instance of the latter, the glycosylase inhibitor can be administered as part of a treatment for a neoplastic conditions, such as in the treatment of leukemias, lymphomas, myelomas, medullomas, medulloblastomas, neuroblastomas, carcinomas, sarcomas and glioblastomas, neoplasias of breast tissue, neoplasias of testicular tissue, neoplasias of endometrial tissue, and neoplasias of testicular tissue, and the like.
In a preferred therapeutic method, the glycosylase inhibitor is contacted with the cell conjointly with a DNA damaging agent. For instance, the DNA damaging agent can include one or more compounds which as DNA alkylating agents and DNA intercalating agents. Exemplary DNA damaging agent include psoralens, tricyclic furocoumarins, dacarbazines, amsacrine, actinomycins, azaserine, bleomycin, carminomycin, daunomycins, mitomycins, mitoxantrones, plicamycins, haloethylnitrosoureas, sulfer mustard, nitrogen mustards, cis-diamminedichloro-platinum(II) (cisplatin), cis-diammino-(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin), cis-diammino-(1,2, -cyclohexyl)-dichloroplatinum(II), cis-(1,2-ethylene-diammine)dichloro-platinum(II), ethylenimines and methylmelamines. In other embodiments, the the DNA damaging agent can include DNA damaging electromagnetic radiation, e.g., ionizing radiation.
The subject method can also be used to inhibit the proliferation of: bacterial cells, e.g., in the treatment of septicemia; fungal cells, e.g., in the treatment of fungicemia; and/or virally-infected cells, e.g., for treating cells infected with a virus having an endogenous glycosylase activity.
Another aspect of the present invention provides a cell (or tissue), e.g., in cell culture, having an impaired DNA repair ability resulting from treatment of the cell with one or more of the subject glycosylase inhibitors. Such cells can be used to ascertain the mutagenic/carcinogenic potential of a chemical or environmental conditions, or can be used to ascertain the ability of a test agent to protect the cell against DNA damage by a known carcinogen/mutagen. In preferred embodiments, the cell is a mammalian cell, more preferably a human cell.
Another aspect of the present invention provides a method for isolating a DNA glycosylase comprising contacting a cytoplasmic preparation of a cell with a solid support (or soluble polymeric support) derivatived with an inhibitor of the present invention, then removing the solid support for contact with the cytoplasmic preparation in order to isolate any glycosylase enzyme bound to the inhibitor.
Yet another aspect of the present invention provides a kit for carrying out conjoint administeration of a glycosylase inhibitor and a DNA damaging agent comprising (i) a DNA damaging agent formulated in a pharmaceutical carrier, and (ii) one or more of the subjecy glycosylase inhibitors, formulated in a pharmaceutical carrier